Nrf2-Mediated Pathway Activated by Prunus spinosa L. (Rosaceae) Fruit Extract: Bioinformatics Analyses and Experimental Validation.

In our previous studies, Prunus spinosa fruit (PSF) ethanol extract was showed to exert antioxidant, antimicrobial, anti-inflammatory and wound healing activities. In the present study, an integrated bioinformatics analysis combined with experimental validation was carried out to investigate the biological mechanism(s) that are responsible for the reported PSF beneficial effects as an antioxidant during a pro-inflammatory TLR4 insult. Bioinformatics analysis using miRNet 2.0 was carried out to address which biological process(es) the extract could be involved in. In addition, Chemprop was employed to identify the key targets of nuclear receptor (NR) signaling and stress response (SR) pathways potentially modulated. The miRNet analysis suggested that the PSF extract mostly activates the biological process of cellular senescence. The Chemprop analysis predicted three possible targets for nine phytochemicals found in the extract: (i) ARE signaling, (ii) mitochondrial membrane potential (MMP) and (iii) p53 SR pathways. The PSF extract antioxidant effect was also experimentally validated in vitro using the human monocyte U937 cell line. Our findings showed that Nrf2 is modulated by the extract with a consequent reduction of the oxidative stress level. This was confirmed by a strong decrease in the amount of reactive oxygen species (ROS) observed in the PSF-treated cells subjected to lipopolysaccharide (LPS) (6 h treatment, 1 µg/mL). No visible effects were observed on p53 and MMP modulation.

Chemical composition, antioxidant and anti-inflammatory properties of Monarda didyma L. essential oil.

In the present study, Monarda didyma L. essential oil (isolated from the flowering aerial parts of the plant) was examined to characterize its chemotype and to evaluate, in addition to the quali-quantitative chemical analysis, the associated antioxidant and anti-inflammatory activities. The plants were grown in central Italy, Urbino (PU), Marche region. Different analyses (TLC, GC-FID, GC-MS and 1H-NMR) allowed the identification of twenty compounds among which carvacrol, p-cymene and thymol were the most abundant. On this basis, the chemotype examined in the present study was indicated as Monarda didyma ct. carvacrol. The antioxidant effect was assessed by DPPH assay. Moreover, this chemotype was investigated for the anti-inflammatory effect in an in vitro setting (i.e., LPS-stimulated U937 cells). The decreased expression of pro-inflammatory cytokine IL-6 and the increased expression of miR-146a are suggestive of the involvement of the Toll-like receptor-4 signaling pathway. Although further studies are needed to better investigate the action mechanism/s underlying the results observed in the experimental setting, our findings show that M. didyma essential oil is rich in bioactive compounds (mainly aromatic monoterpenes and phenolic monoterpenes) which are most likely responsible for its beneficial effect.

Do ancient wheats contain less gluten than modern bread wheat, in favour of better health?

Popular media messaging has led to increased public perception that gluten-containing foods are bad for health. In parallel, 'ancient grains' have been promoted with claims that they contain less gluten. There appears to be no clear definition of 'ancient grains' but the term usually includes einkorn, emmer, spelt and Khorasan wheat. Gluten is present in all wheat grains and all can induce coeliac disease (CD) in genetically susceptible individuals. Analyses of 'ancient' and 'modern' wheats show that the protein content of modern bread wheat (Triticum aestivum) has decreased over time while the starch content increased. In addition, it was shown that, compared to bread wheat, ancient wheats contain more protein and gluten and greater contents of many CD-active epitopes. Consequently, no single wheat type can be recommended as better for reducing the risks of or mitigating the severity of CD. An estimated 10% of the population of Western countries suffers from gastrointestinal symptoms that lack a clear organic cause and is often referred to as irritable bowel syndrome (IBS). Many of these patients consider themselves gluten sensitive, but in most cases this is not confirmed when tested in a medical setting. Instead, it may be caused by gas formation due to fermentation of fructans present in wheat or, in some patients, effects of non-gluten proteins. A significant overlap of symptoms with those of CD, IBS and inflammatory bowel disease makes a medical diagnosis a priority. This critical narrative review examines the suggestion that 'ancient' wheat types are preferred for health and better tolerance.

Wheat amylase/trypsin inhibitors (ATIs): occurrence, function and health aspects.

Amylase/trypsin inhibitors (ATIs) are widely consumed in cereal-based foods and have been implicated in adverse reactions to wheat exposure, such as respiratory and food allergy, and intestinal responses associated with coeliac disease and non-coeliac wheat sensitivity. ATIs occur in multiple isoforms which differ in the amounts present in different types of wheat (including ancient and modern ones). Measuring ATIs and their isoforms is an analytical challenge as is their isolation for use in studies addressing their potential effects on the human body. ATI isoforms differ in their spectrum of bioactive effects in the human gastrointestinal (GI), which may include enzyme inhibition, inflammation and immune responses and of which much is not known. Similarly, although modifications during food processing (exposure to heat, moisture, salt, acid, fermentation) may affect their structure and activity as shown in vitro, it is important to relate these changes to effects that may present in the GI tract. Finally, much of our knowledge of their potential biological effects is based on studies in vitro and in animal models. Validation by human studies using processed foods as commonly consumed is warranted. We conclude that more detailed understanding of these factors may allow the effects of ATIs on human health to be better understood and when possible, to be ameliorated, for example by innovative food processing. We therefore review in short our current knowledge of these proteins, focusing on features which relate to their biological activity and identifying gaps in our knowledge and research priorities.

Effects of Animal-Assisted Therapy (AAT) in Alzheimer's Disease: A Case Study.

Alzheimer's disease (AD) is a neurodegenerative disorder, characterized by cortical dementia and irreversibly progressive developments leading to a vegetative state and, finally, to death. Although many aspects of its etiology, diagnosis and treatment still remain obscure and the current approach to the disease mostly suffers from limited and low-efficiency therapeutic means, nevertheless, recent interventions have aimed at improving patients' quality of life through nonpharmacological approaches, including animal-assisted therapy (AAT), arousing growing interest. In order to assess the physiological and neuropsychological effects of AAT on AD, 24 residents of a rest house in northern Italy were enrolled. The intervention consisted of one 45-minute AAT session per week over ten weeks. Twelve residents (six AD and six non-AD) received AAT and twelve (six AD and six non-AD) were controls. In order to evaluate the physiological and clinical effect of AAT on AD residents, three cardiac parameters, including the systolic and diastolic blood pressure and heart rate, were measured. Moreover, the neurocognitive and depressive states were assessed by the Mini Mental State Examination and the Geriatric Depression Scale, respectively. Analyses were performed by a four-way ANOVA model (including two ways for repeated measures) considering each main effect and interaction possible in the design. Our findings, despite the small sample size, suggest that AAT has a positive significant effect on physiological parameters and neurocognitive impairment, while no effect was observed on the depression level.

Molecular and biometric data on Carabus (Macrothorax) morbillosus Fabricius, 1792 (Coleoptera, Carabidae) from Mid Mediterranean areas.

The present study was carried out using molecular and biometric data of Carabus (Macrothorax) morbillosus from mid-Mediterranean areas to determine additional information on basal relationships among its representative subspecies. To this aim, two different kinds of approach were employed, including a morphometric analysis of four morphological parameters (i.e., elytra length, elytra width, pronotum length, pronotum width) of 128 specimens, and a Bayesian genetic analysis of 44 cytochrome oxidase subunit I (COI) partial sequences (i.e., 38 examined for the first time and six retrieved from GenBank database). Representative populations of C. (M.) morbillosus were sampled in four countries, namely Italy, Malta, Spain, and Tunisia. The present findings support the validity of four C. (M.) morbillosus subspecies, specifically C. (M.) m. alternans, C. (M.) m. bruttianus, C. (M.) m. constantinus, and C. (M.) m. macilentus, and redefine these subspecies' distributions. Notably, within the C. (M.) m. constantinus clade, two (i.e., Sardinia/Tuscany and Lampedusa) out of the three subgroups appear as homogeneous geographical groupings.

Molecular analysis of Lepidopleuruscajetanus (Poli, 1791) (Polyplacophora, Leptochitonidae) from the Mediterranean and near Atlantic.

In the present paper we used a molecular data set (including mitochondrial partial 16S rRNA and COI gene sequences) to examine the genetic structure of Lepidopleuruscajetanus (Poli, 1791) (Polyplacophora, Leptochitonidae) - a distinctive shallow water chiton and member of the basal branching Lepidopleurida, which is widespread in and adjacent to the Mediterranean. The analyses of the two mt-standard marker fragments resolved two main discrete clusters reported as L.cajetanus s.s. and L.aff.cajetanus, respectively. Lepidopleuruscajetanus s.s. is widespread throughout the area under study, while the second distinct lineage apparently co-occurs on the eastern Spanish mainland coast of the Balearic Sea. This result is discussed comparing our data with those reported, in 2014, by Fernández and colleagues who described L.cajetanus as exhibiting "a 'chaotic patchiness' pattern defined by a high genetic variability with locality-exclusive haplotypes, high genetic divergence, and a lack of geographic structure". Although genetic data alone are not sufficient to draw any definitive conclusions, nevertheless we believe that present results shed new light on L.cajetanus which apparently shows more geographically patterned genetic structure than supposed so far.

Antioxidant and Anti-Inflammaging Ability of Prune (Prunus Spinosa L.) Extract Result in Improved Wound Healing Efficacy.

Prunus spinosa L. fruit (PSF) ethanol extract, showing a peculiar content of biologically active molecules (polyphenols), was investigated for its wound healing capacity, a typical feature that declines during aging and is negatively affected by the persistence of inflammation and oxidative stress. To this aim, first, PSF anti-inflammatory properties were tested on young and senescent LPS-treated human umbilical vein endothelial cells (HUVECs). As a result, PSF treatment increased miR-146a and decreased IRAK-1 and IL-6 expression levels. In addition, the PSF antioxidant effect was validated in vitro with DPPH assay and confirmed by in vivo treatments in C. elegans. Our findings showed beneficial effects on worms' lifespan and healthspan with positive outcomes on longevity markers (i.e., miR-124 upregulation and miR-39 downregulation) as well. The PSF effect on wound healing was tested using the same cells and experimental conditions employed to investigate PSF antioxidant and anti-inflammaging ability. PSF treatment resulted in a significant improvement of wound healing closure (ca. 70%), through cell migration, both in young and older cells, associated to a downregulation of inflammation markers. In conclusion, PSF extract antioxidant and anti-inflammaging abilities result in improved wound healing capacity, thus suggesting that PSF might be helpful to improve the quality of life for its beneficial health effects.

Prunus spinosa Extract Loaded in Biomimetic Nanoparticles Evokes In Vitro Anti-Inflammatory and Wound Healing Activities.

Prunus spinosa fruits (PSF) contain different phenolic compounds showing antioxidant and anti-inflammatory activities. Innovative drug delivery systems such as biomimetic nanoparticles could improve the activity of PSF extract by promoting (i) the protection of payload into the lipidic bilayer, (ii) increased accumulation to the diseased tissue due to specific targeting properties, (iii) improved biocompatibility, (iv) low toxicity and increased bioavailability. Using membrane proteins extracted from human monocyte cell line THP-1 cells and a mixture of phospholipids, we formulated two types of PSF-extract-loaded biomimetic vesicles differing from each other for the presence of either 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG). The biological activity of free extract (PSF), compared to both types of extract-loaded vesicles (PSF-DOPCs and PSF-DOPGs) and empty vesicles (DOPCs and DOPGs), was evaluated in vitro on HUVEC cells. PSF-DOPCs showed preferential incorporation of the extract. When enriched into the nanovesicles, the extract showed a significantly increased anti-inflammatory activity, and a pronounced wound-healing effect (with PSF-DOPCs more efficient than PSF-DOPG) compared to free PSF. This innovative drug delivery system, combining nutraceutical active ingredients into a biomimetic formulation, represents a possible adjuvant therapy for the treatment of wound healing. This nanoplatform could be useful for the encapsulation/enrichment of other nutraceutical products with short stability and low bioavailability.

Bioeffects of Prunus spinosa L. fruit ethanol extract on reproduction and phenotypic plasticity of Trichoplax adhaerens Schulze, 1883 (Placozoa).

The aim of this work was to test and analyse the bioeffects of Prunus spinosa L. (Rosacaee) fruit ethanol extract on Trichoplax adhaerens Schulze, 1883 (Placozoa) laboratory cultures which-for the first time-were employed as in vivo biological model to assess the bioactivity of a natural extract. The ethanol extract of P. spinosa was administrated during a 46 day experimental period; ultrastructural (by optical, confocal, TEM and SEM microscopy) and morphometric analyses indicated that treated Trichoplax adhaerens showed significant differences in viability, reproductive modalities, body shape and colour with respect to the control group. Finally, P. spinosa bioactive compounds seem to exert profound protective effects on T. adhaerens reproduction and phenotype. Our results may support additional investigations related to other bioactive compounds properties useful for nutraceutical preparations to be used as food supplements.

Sex-Biased Dispersal Obscures Species Boundaries in Integrative Species Delimitation Approaches.

Accurate delimitation of species is crucial for a stable taxonomy, which provides the foundation for the study of evolutionary biology, ecology, and essentially all biological disciplines. Several approaches toward impartial and repeatable taxonomic practices are available but all existing methods have potentially unacceptable shortcomings. In particular, problems can arise when the underlying model assumptions are violated, for instance, in the presence of reduced gene flow. This is observed in the context of sex-biased dispersal, which is a common but underappreciated feature in many groups of organisms. Previously, simulations have indicated that sex-biased dispersal may lead to erroneous estimations of the true species numbers. However, this phenomenon has never been examined using empirical data. We evaluate the bias introduced by extreme female philopatry on a range of de novo [GMYC, PTP, ABGD, statistical parsimony, trinomial distribution of triplets model (tr2)] and validation (STACEY, iBPP) approaches to species delimitation in the scarab beetle genus Pachypus. Since female philopatry exhibited in this genus in particular can affect mitochondrial gene flow, we compared the results from analyses of single loci, mitochondrial loci, nuclear loci and combined data, as well as the performance of morphometric data as a secondary data source in a fully integrative Bayesian framework. Large overestimation of species numbers was observed across all analyses of combined and mitochondrial DNA data sets, suggesting specimens from nearly every sampling location as separate species. The use of nuclear data resulted in more reasonable estimations of species boundaries, which were largely supported by morphometrics of linear measurements, while geometric morphometrics of body outlines resulted in stronger splitting. Simulations of population divergence with migration, corresponding to the biology of Pachypus, showed that female philopatry strongly increases reciprocal monophyly of mitochondrial markers and may substantially contribute to over-splitting in species delimitation. Robust results recovered using nuclear DNA and morphological data nevertheless enabled us to reach novel conclusions about species boundaries in Pachypus. Our findings suggest that mitochondrial DNA will be less suited to species delimitation in many cases, in particular in the presence of sex-biased dispersal.

MiR-126 in intestinal-type sinonasal adenocarcinomas: exosomal transfer of MiR-126 promotes anti-tumour responses.

BACKGROUND: Intestinal-type sinonasal adenocarcinomas (ITACs) are aggressive malignancies related to wood dust and leather exposure. ITACs are generally associated with advanced stage at presentation due to the insidious growth pattern and non-specific symptoms. Therefore, biomarkers that can detect the switch from the benign disease to malignancy are needed. Essential for tumour growth, angiogenesis is an important step in tumour development and progression. This process is strictly regulated, and MiR-126 considered its master modulator. METHODS: We have investigated MiR-126 levels in ITACs and compared them to benign sinonasal lesions, such as sinonasal-inverted papillomas (SIPs) and inflammatory polyps (NIPs). The tumour-suppressive functions of MiR-126 were also evaluated. RESULTS: We found that MiR-126 can significantly distinguish malignancy from benign nasal forms. The low levels of MiR-126 in ITACs point to its role in tumour progression. In this context, restoration of MiR-126 induced metabolic changes, and inhibited cell growth and the tumorigenic potential of MNSC cells. CONCLUSIONS: We report that MiR-126 delivered via exosomes from endothelial cells promotes anti-tumour responses. This paracrine transfer of MiRs may represent a new approach towards MiR-based therapy.

Evaluation of the safety of ancient strains of wheat in coeliac disease reveals heterogeneous small intestinal T cell responses suggestive of coeliac toxicity.

BACKGROUND & AIMS: Coeliac disease is a chronic small intestinal immune-mediated enteropathy triggered by dietary gluten in genetically predisposed individuals. Since it is unknown if all wheat varieties are equally toxic to coeliac patients seven Triticum accessions showing different origin (ancient/modern) and ploidy (di-, tetra- hexaploid) were studied. MATERIALS AND METHODS: Selected strains of wheat were ancient Triticum monococcum precoce (AA genome) and Triticum speltoides (BB genome), accessions of Triticum turgidum durum (AABB genome) including two ancient (Graziella Ra and Kamut) and two modern (Senatore Cappelli and Svevo) durum strains of wheat and Triticum aestivum compactum (AABBDD genome). Small intestinal gluten-specific T-cell lines generated from 13 coeliac patients were tested with wheat accessions by proliferation assays. RESULTS: All strains of wheat independent of ploidy or ancient/modern origin triggered heterogeneous responses covering wide ranges of stimulation indices. CONCLUSION: Ancient strains of wheat, although previously suggested to be low or devoid of coeliac toxicity, should be tested for immunogenicity using gluten-specific T-cell lines from multiple coeliac patients rather than gluten-specific clones to assess their potential toxicity. Our findings provide further evidence for the need for a strict gluten-free diet in coeliac patients, including avoidance of ancient strains of wheat.

Immunogenicity characterization of two ancient wheat α-gliadin peptides related to coeliac disease.

The immunogenic potential of α-gliadin protein from two ancient wheats was studied with reference to coeliac disease. To this aim we investigated Graziella Ra® and Kamut® (the latter is considered an ancient relative of modern durum wheat) in comparison to four durum wheat accessions (Senatore Cappelli, Flaminio, Grazia and Svevo). ELISA and Western Blot analyses - carried out by two monoclonal antibodies raised against the α-gliadin peptides p31-49 (LGQQQPFPQQPYPQPQPF) and p56-75 (LQLQPFPQPQLPYPQPQLPY) containing a core region (underlined) reported to be toxic for coeliac patients - always showed an antibody-antigen positive reaction. For all accessions, an α-gliadin gene has also been cloned and sequenced. Deduced amino acid sequences constantly showed the toxic motifs. In conclusion, we strongly recommend that coeliac patients should avoid consuming Graziella Ra® or Kamut®. In fact their α-gliadin not only is as toxic as one of the other wheat accessions, but also occurs in greater amount, which is in line with the higher level of proteins in ancient wheats when compared to modern varieties.

Heterochromatin of the scarab beetle, Bubas bison (Coleoptera: Scarabaeidae) II. Evidence for AT-rich compartmentalization and a high amount of rDNA copies.

An unexpected result arising from a previous characterization of the scarab beetle Bubas bison (Coleoptera: Scarabaeidae) heterochromatin was its unusual homogeneous reaction to different staining methods. In particular, silver stainability of heterochromatic ends of all chromosomes prevented identification of the number of rDNA transcriptionally active regions. Data formerly obtained using silver impregnation (Ag-NOR), C- G- and DAPI banding are here improved and completed by application of CMA(3) staining and rDNA FISH with the aim to investigate heterochromatin base composition and locate rDNA regions with respect to NOR-associated heterochromatin. Our results show that B. bison has a high amount of heterochromatin (almost 50%) and that--as revealed by rDNA FISH--major rRNA genes are spread over the heterochromatic telomeric regions of eight chromosomes, thus suggesting that only a portion, although consistent, of total heterochromatin is associated with ribosomal clusters. Moreover, DAPI-positive (AT-specific) and CMA(3)-negative (GC-specific) reactions of heterochromatic DNA confirm its AT-rich composition. Finally, possible explanations for the bright DAPI-fluorescence of both heterochromatin and rDNA sequences are discussed.

Cytogenetics of the land snails Cantareus aspersus and C. mazzullii (Mollusca: Gastropoda: Pulmonata).

A cytogenetic study was carried out on the chromosomes and nuclear DNA contents of the land snails Cantareus aspersus and C. mazzullii (Gastropoda: Pulmonata). Chromosomes were studied using Giemsa staining, banding methods and fluorescent in situ hybridization (FISH) with three repetitive DNA probes [18S rDNA, (GATA)(n) and (TTAGGG)(n)]. Results were very similar in the two species both showing (1) 54 bi-armed chromosomes [submetacentrics (SM) + metacentrics (M) + subtelocentrics (ST)]; (2) 10 terminal NORs after sequential application of rDNA FISH and silver staining; (3) uniform DNA fluorescence with CMA(3) and DAPI staining and (4) genomic composition considerably enriched both in highly- and moderately-repeated DNAs. The telomeric (TTAGGG)(n) sequence hybridized with the termini of all of the chromosomes in the two species. In spite of their apparent karyological uniformity, flow cytometry DNA assays showed that C. aspersus and C. mazzullii are characterized by different nuclear DNA content (C values are 3.58 and 3.08 pg, respectively) and slightly different base composition in their genomes. Present data on GS and AT% in C. mazzullii and C. aspersus confirm the trend toward high GS values and GC percentages among land snails.

Chromosomal location polymorphism of major rDNA sites in two Mediterranean populations of the killifish Aphanius fasciatus (Pisces: Cyprinodontidae).

The chromosomes of the Mediterranean killifish, Aphanius fasciatus from two populations, the Lagoon of Venice (LV, 15 specimens) and the Lagoon "Stagnone di Marsala" (Sicily) (SM, 48 specimens), have been investigated using conventional Ag-staining and fluorescent in situ hybridization (FISH) with 18S rDNA probe. The two methods revealed variation in the number of major rDNA sites ranging from 8 to 14 (LV) and from 1 to 4 (SM) per individual. The fact that each individual possessed its own number of sites implies that observed variation was structural. Moreover, overlapping of silver staining and FISH patterns demonstrated that all ribosomal genes were transcriptionally active in each specimen.

Karyotype, banding and rDNA FISH in the scarab beetle Anoplotrupes stercorosus (Coleoptera Scarabaeoidea: Geotrupidae). Description and comparative analysis.

Six specimens of Anoplotrupes stercorosus (Coleoptera Scarabaeoidea: Geotrupidae) were analysed using conventional staining, banding techniques and fluorescent in situ hybridization with a ribosomal probe (rDNA FISH). Detailed karyotype description was also joined to a comparative analysis between present data and those previously reported for Thorectes intermedius [Chromosome Res. 7 (1999) 1]. The two species, both belonging to the tribe Geotrupini, show the same modal number but different autosomal morphology which is in contrast with the high chromosome stability argued for Geotrupinae during the last three decades. Moreover, a detailed comparison reveals the occurrence of a plesiomorphic condition in A. stercorosus with respect to the apomorphic one of T. intermedius. This finding agrees with phylogenetic relationships proposed for the two genera based on morphological and anatomical characters.

Evidence for male XO sex-chromosome system in Pentodon bidens punctatum (Coleoptera Scarabaeoidea: Scarabaeidae) with X-linked 18S-28S rDNA clusters.

In scarab beetle species of the genus Pentodon, the lack of analysis of sex chromosomes in females along with the poor characterization of sex chromosomes in the males, prevented all previous investigations from conclusively stating sex determination system. In this study, somatic chromosomes from females and spermatogonial chromosomes from males of Pentodon bidens punctatum (Coleoptera: Scarabaeoidea: Scarabaeidae) from Sicily have been analyzed using non-differential Giemsa staining. Two modal numbers of chromosomes were obtained: 2n = 20 and 19 in females and males, respectively. This finding along with other karyological characteristics such as the occurrence of one unpaired, heterotypic chromosome at metaphase-I and two types of metaphase-II spreads in spermatocytes demonstrate that a XO male/XX female sex determining mechanism - quite unusual among Scarabaeoidea - operates in the species investigated here. Spermatocyte chromosomes have also been examined after a number of banding techniques and fluorescent in situ hybridization with ribosomal sequences as a probe (rDNA FISH). The results obtained showed that silver and CMA(3) staining were inadequate to localize the chromosome sites of nucleolus organizer regions (NORs) due to the over-all stainability of both constitutive heterochromatin and heterochromatin associated to the NORs. This suggests that heterochromatic DNA of P. b. punctatum is peculiar as compared with other types of heterochromatin studied so far in other invertebrate taxa. By rDNA FISH major ribosomal genes were mapped on the X chromosome.